WO2019098361A1 - 多能性幹細胞を用いた自殺遺伝子脳腫瘍治療薬 - Google Patents

多能性幹細胞を用いた自殺遺伝子脳腫瘍治療薬 Download PDF

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WO2019098361A1
WO2019098361A1 PCT/JP2018/042629 JP2018042629W WO2019098361A1 WO 2019098361 A1 WO2019098361 A1 WO 2019098361A1 JP 2018042629 W JP2018042629 W JP 2018042629W WO 2019098361 A1 WO2019098361 A1 WO 2019098361A1
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gene
stem cells
cell
cells
neural stem
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French (fr)
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戸田　正博
岡野　栄之
浩之 三好
亮太 田村
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学校法人 慶應義塾
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Priority to JP2019554440A priority Critical patent/JP7150233B2/ja
Priority to US16/765,257 priority patent/US12090172B2/en
Priority to EP18878961.4A priority patent/EP3714894B1/de
Priority to CN201880074333.5A priority patent/CN111356463A/zh
Publication of WO2019098361A1 publication Critical patent/WO2019098361A1/ja
Priority to JP2022083217A priority patent/JP2022103424A/ja

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    • A—HUMAN NECESSITIES
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
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    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N9/10—Transferases (2.)
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    • C12Y204/00—Glycosyltransferases (2.4)
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    • C12Y204/02009—Uracil phosphoribosyltransferase (2.4.2.9)
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    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • the present invention relates to a cell preparation for treating a brain tumor, and a method for producing neural stem cells used in the cell preparation.
  • BTSCs brain tumor stem cells
  • BTSCs brain tumor stem cells
  • Suicide gene therapy using a viral vector was expected to be effective because it exerts a bystander effect, but the spread to invading tumor cells was insufficient, so the results of clinical trials were limited .
  • neural stem cells NSCs
  • NSCs neural stem cells
  • attempts to apply neural stem cells as a cell vehicle to suicide gene therapy are attracting attention.
  • technology related to suicide gene therapy using mesenchymal stem cells having properties similar to neural stem cells has also been developed (Patent Document 1).
  • neural stem cells As described above, although there is great hope for suicide gene therapy using neural stem cells and mesenchymal stem cells, obtaining clinically sufficient human doses of neural stem cells in clinical practice is an ethical issue Is difficult. Mesenchymal stem cells are also easier to obtain than neural stem cells, but their collection amount is limited as long as they are collected from the living body.
  • herpes simplex virus thymidine kinase gene HSVtk
  • HSVtk herpes simplex virus thymidine kinase gene
  • CD cytosine deaminase
  • UPRT uracil phosphoribosyltransferase
  • the present invention solves the problem of unavailability in neural stem cells and mesenchymal stem cells, and provides a new therapeutic means for treating brain tumors using the CD-UPRT gene having high antitumor effect.
  • the purpose is to
  • the present inventor has found that by inducing differentiation of neural stem cells from iPS cells, neural stem cells sufficient for use in suicide gene therapy can be obtained. .
  • the CD-UPRT gene into the housekeeping gene region of iPS cells by genome editing, it is possible to obtain a neural stem cell expressing CD-UPRT gene excellent in terms of safety, quality control, and stable supply. Found out.
  • this CD-UPRT gene-expressing neural stem cell exhibits an antitumor effect not only on brain tumor cells but also on brain tumor stem cells.
  • the present invention has been completed based on the above findings. That is, the present invention provides the following [1] to [17].
  • a cell preparation for treating a brain tumor which is used together with a prodrug converted to 5-fluorouracil by cytosine deaminase, comprising neural stem cells derived from pluripotent stem cells having a cytosine deaminase gene and a uracil phosphoribosyltransferase gene
  • a cell preparation for treating a brain tumor characterized by
  • a pluripotent stem cell-derived neural stem cell is a neural stem cell obtained by performing the following step (1) and then performing the step (2) [1] or [2] The cell preparation for treating a brain tumor according to (1) introducing cytosine deaminase gene and uracil phosphoribosyltransferase gene into pluripotent stem cells; (2) A step of differentiating pluripotent stem cells into neural stem cells.
  • the pluripotent stem cell-derived neural stem cell is a neural stem cell obtained by performing the following step (1) and then performing step (2) [1] or [2] The cell preparation for treating a brain tumor according to (1) differentiating pluripotent stem cells into neural stem cells (2) A step of introducing a cytosine deaminase gene and a uracil phosphoribosyltransferase gene into neural stem cells.
  • the present invention provides a novel therapeutic means for brain tumors using neural stem cells derived from pluripotent stem cells such as iPS cells and ES cells.
  • the upper stage and the interruption show a treatment group, and the lower stage shows a control group.
  • Images are, from the left, Venus staining (staining U87 tumor cells), KO staining (staining TSC), DAPI staining (staining nucleus), merge (fused image of Venus, KO, DAPI).
  • TSC yCD-UPRT expression / treatment stem cells
  • IVIS luminescence imaging analysis
  • Photographs of brains removed from mice left), HE stained images of mouse brain sections (middle), and Venus fluorescence images of mouse brain sections (right).
  • the upper row shows the treatment group, and the lower row shows the control group. Images are, from the left, Venus staining (staining U87 tumor cells), KO staining (staining TSC), DAPI staining (staining nucleus), merge (fused image of Venus, KO, DAPI).
  • the figure which shows the result of survival analysis of a brain tumor model The figure which shows the result of the time-lapse imaging of yCD-UPRT expression TSC.
  • the upper part shows a treatment group
  • the middle part shows a control group 1
  • the lower part shows a control group 2.
  • KO stained image of yCD-UPRT expressing TSC The upper row shows the treatment group, and the lower row shows the control group 1.
  • the upper row shows the treatment group, and the lower row shows the control group 1.
  • the cell preparation for treating a brain tumor of the present invention is a cell preparation for treating a brain tumor which is used together with a prodrug which is converted to 5-fluorouracil by cytosine deaminase (CD), which comprises a cytosine deaminase gene and a uracil phosphoribosyltransferase gene (CD- It is characterized in that it comprises neural stem cells derived from pluripotent stem cells having UPRT gene).
  • CD cytosine deaminase
  • pluripotent stem cells in the present invention can be interpreted in the meaning generally used by those skilled in the art, and includes, for example, iPS cells and ES cells.
  • brain tumor means a tumor that develops in intracranial tissue, and specific examples thereof include glioma (glioma), medulloblastoma, neuroblastoma, meningioma, pituitary adenoma, There may be mentioned schwannoma, primary central nervous system lymphoma, sarcoma, spinal cord tumor and so on. In the present invention, any of these brain tumors can be treated, but gliomas are preferably treated.
  • treatment is meant to include not only killing tumor cells, but also reducing tumor cells and inhibiting the growth of tumor cells.
  • the CD gene and the UPRT gene are suicide genes having 5-fluorocytosine (5-FC) and 5-fluorouracil (5-FU) as prodrugs, respectively.
  • Vectors expressing these suicide genes are commercially available, and such vectors can be used to produce pluripotent stem cells and neural stem cells expressing these suicide genes.
  • the CD gene and the UPRT gene each have an anti-tumor effect, but introducing these two genes into cells provides about 100 times the anti-tumor effect when the CD gene is introduced alone. Are known.
  • the prodrug in the present invention may be anything that is converted to 5-FU by CD.
  • Specific examples of such prodrugs include, but are not limited to, 5-FC.
  • neural stem cells in the present invention refer to stem cells having the ability to supply cells that differentiate into neurons and glial cells.
  • the cell preparation of the present invention contains the neural stem cells, but may contain cells other than neural stem cells as long as the therapeutic effect of the brain tumor is not adversely affected.
  • neural stem cells are produced from iPS cells according to the method described in Mol Brain 9: 85, 2016 described later, not only neural stem cells but also neural progenitor cells are generated.
  • the cell preparation of the present invention may include both neural stem cells and neural progenitor cells.
  • neural stem cells used in the present invention are neural stem cells derived from pluripotent stem cells such as iPS cells, and neural stem cells collected from the brain or the like are not used as they are.
  • the pluripotent stem cell-derived neural stem cell having the CD-UPRT gene is subjected to the step (A1) described below and then to the step (A2), or the step (B1) is performed, and then the step (B2) It can be produced by the method to be performed.
  • the CD-UPRT gene is introduced into pluripotent stem cells.
  • ES cells can also be used, but iPS cells are preferably used.
  • the iPS cells to be used are not particularly limited as long as they can be differentiated into neural stem cells, from their origin, reprogramming factors to be introduced, methods for introducing reprogramming factors, etc.
  • the cell preparation of the present invention is mainly human. In this case, it is preferable to use human-derived iPS cells because they are used for treatment of brain tumors.
  • iPS cells iPS cells derived from patients to which a cell preparation is administered may be used, or iPS cells derived from humans other than patients may be used.
  • the method for introducing a reprogramming factor is not limited, but it is preferable to use an integration-free iPS cell.
  • the iPS cells to be used may be prepared according to known methods, but can also be obtained from research institutes such as Kyoto University iPS Cell Research Institute (CiRA).
  • the introduction of the CD-UPRT gene into pluripotent stem cells may be performed using a viral vector, but is preferably performed using genome editing. This is because, when a suicide gene is inserted into the genome of pluripotent stem cells using a virus vector such as lentiviral vector, the suicide gene is randomly inserted into the chromosome, so mutation of the insertion site and activation of peripheral genes , There is a concern about inactivation of suicide gene by position effect. It is thought that such problems can be avoided by using genome editing rather than viral vectors.
  • Genome editing can be performed using ZFN, TALEN, CRISPR / Cas9 and the like, and among these, it is preferable to use CRISPR / Cas9. It is preferable to insert the CD-UPRT gene into the region of the housekeeping gene of pluripotent stem cells by genome editing.
  • the housekeeping gene is not particularly limited as long as it is expressed in fixed amounts in many cells. Specific examples thereof include glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, cyclophilin gene, ⁇ -actin gene, ⁇ -tubulin gene and the like. Among these, GAPDH gene is preferable.
  • the CD-UPRT gene may be inserted into the AAVS1 region of pluripotent stem cells.
  • Adeno-associated virus (AAV) is not pathogenic to humans, and the AAVS1 region is considered as a highly safe genomic region for insertion of foreign genes.
  • the CD-UPRT gene When the CD-UPRT gene is introduced into pluripotent stem cells using a viral vector, as described above, the CD-UPRT gene can not be expressed, as it may be adversely affected by random insertion into the chromosome. It is preferable to use a gene construct that can be artificially regulated.
  • a gene construct can be prepared using a known expression induction system, a tetracycline induction system (PLoS One 8: e59890, 2013). This tetracycline inducible system consists of a region that expresses reverse tetracycline regulatory transactivator (rtTA), and a region that contains the gene of interest and the TRE promoter located upstream of it.
  • rtTA reverse tetracycline regulatory transactivator
  • rtTA By binding to tetracycline, rtTA binds to the TRE promoter and induces the expression of a target gene located downstream.
  • the expression of a suicide gene can be induced by the addition of tetracycline.
  • the induction of expression in this system can also be performed with tetracycline as described above, but is usually performed with doxycycline, which is a derivative of tetracycline.
  • a gene construct capable of artificially regulating the expression of the CD-UPRT gene can also be produced using various inducible promoters such as drug-inducible promoters, in addition to this tetracycline inducible system.
  • viral vectors examples include adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors and the like. Among these, vectors of the type that integrate genes into the genome are preferred, and lentiviral vectors are most preferred.
  • the vector may contain a sequence such as a reporter gene or IRES.
  • step (A2) pluripotent stem cells are differentiated into neural stem cells.
  • Differentiation from pluripotent stem cells to neural stem cells may be performed according to an embryoid body-mediated method, a non-embryoid body-mediated method, or any known method.
  • it can be performed according to the method described in Stem Cell Reports. 2017 Nov 14; 9 (5): 1675-1691.
  • formation of embryoid bodies from iPS cells can be performed using a known embryoid body formation medium, and the embryoid body medium contains TGF ⁇ family inhibitor (eg, SB431542) and BMP An inhibitor (eg, LDN-193189) is included.
  • the neurosphere medium contains epidermal growth factor, fibroblast growth factor-2, leukemia inhibitory factor, B-27 supplement and the like.
  • the neurosphere medium contains epidermal growth factor, fibroblast growth factor-2, leukemia inhibitory factor, B-27 supplement and the like.
  • a cell mass containing neural stem cells called neurospheres is formed.
  • the formed neurospheres are recovered, dispersed into single cells, and cultured in neurosphere medium to form neurospheres again. After repeating such operation several times, neural stem cells can be obtained by recovering the neurosphere.
  • pluripotent stem cell-derived neural stem cells having a CD-UPRT gene When pluripotent stem cell-derived neural stem cells having a CD-UPRT gene are produced by sequentially performing steps (A1) and (A2), neural stem cells for therapeutic use can be stably produced by proliferating pluripotent stem cells. It has the advantage of being able to provide.
  • step (B1) pluripotent stem cells are differentiated into neural stem cells.
  • the differentiation from pluripotent stem cells to neural stem cells in step (B1) can be performed in the same manner as the differentiation in step (A2).
  • the CD-UPRT gene is introduced into neural stem cells.
  • step (B2) The introduction of the CD-UPRT gene into neural stem cells in step (B2) can be performed in the same manner as the introduction in step (A1).
  • step (A1) it is preferable to use genome editing or to make the CD-UPRT gene a gene construct capable of artificially regulating the expression, since there may be a problem of cytotoxicity. It is usually not necessary to do these things, since in step (B2) the possibility of cytotoxicity problems is very low.
  • the "object” called “neural stem cell” is specified not by the structure or the characteristic but by the manufacturing method, because the cell is a part of the living body Because their structure and characteristics are extremely complex, and their task of identifying them requires a significant amount of economic expenditure and time.
  • the cell preparation of the present invention may contain, in addition to neural stem cells, other pharmaceutically acceptable components.
  • Such other components may include carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, physiological saline and the like.
  • dimethylsulfoxide or serum albumin may be contained to protect cells at the time of cryopreservation, and an antibiotic may be contained to prevent bacterial contamination and growth.
  • the number of neural stem cells contained in the cell preparation of the present invention is the sex, age, weight, and affected area of the subject so as to obtain the desired effect (eg, disappearance of tumor, reduction of tumor size) in the treatment of brain tumor. It can be determined appropriately in consideration of the condition, the condition of the cells to be used, and the like.
  • the cell preparation of the present invention can be administered several times (for example, 2 to 10 times), at intervals (for example, twice a day, once a day, twice a week, once a week, once a two weeks Times) may be administered.
  • the dose can be appropriately determined in consideration of the sex, age, body weight, condition of affected area, condition of cells used, etc., but 1 ⁇ 10 6 to 1 ⁇ 10 6 stem cells per individual (human) Ten doses of 1-10 doses are preferred.
  • the administration site and administration method of the cell preparation of the present invention are not particularly limited. Examples of administration methods include tumor local administration, intracarotid administration, intravenous administration and the like.
  • the administration site and administration method of the prodrug used together with the cell preparation of the present invention are not particularly limited.
  • a method of administration in addition to the above-mentioned tumor local administration, intracarotid administration, intravenous administration, intraperitoneal administration and the like can be mentioned.
  • the administration time of the prodrug may be before, simultaneously with, or after administration of the cell preparation of the present invention, but usually, it is divided into multiple doses after administration of the cell preparation.
  • the dose of the prodrug can be appropriately determined in consideration of the type of prodrug used, the sex, age, body weight of the subject, the condition of the affected area, etc., but in the case of administering 5-FC, one individual A dose of 50 to 200 mg / kg, four times a day for 2 to 3 weeks (28 to 42 times) is preferable once per (human).
  • doxycycline will also be given at the time of treatment to express the suicide gene.
  • administration methods of doxycycline include oral administration, tumor local administration, intracarotid administration, intravenous administration, intraperitoneal administration and the like.
  • Doxycycline may be administered before, simultaneously with, or after administration of the cell preparation of the present invention, but is usually administered after administration of the cell preparation.
  • Example 1 Suicide gene cell therapy for glioma cells
  • the human iPS cells (1210B2) used were obtained from Kyoto University iPS Cell Research Institute (CiRA). 1210 B2 is a method for introducing a reprogramming factor into human peripheral blood mononuclear cells using an episomal vector (Okita K, Yamakawa T, Matsumura Y, Sato Y, Amano N, Watanabe A, Goshima N, Yamanaka S. An efficient It was established by nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells. 2013 Mar; 31 (3): 458-66.
  • Human iPS cells are seeded on a plastic culture dish coated with iMatrix-511 (manufactured by Nippi), and a known feeder-free method (Nakagawa M, Taniguchi Y) using Stem Fit AK03 or AK03N medium (manufactured by Ajinomoto Co., Ltd.).
  • a known feeder-free method (Nakagawa M, Taniguchi Y) using Stem Fit AK03 or AK03N medium (manufactured by Ajinomoto Co., Ltd.).
  • Senda S Takizawa N, Ichisaka T, Asano K, Morizane A, Doi D, Takahashi J, Nishizawa M, Yoshida Y, Toyoda T, Osafune K, Sekiguchi K, Yamanaka S.
  • a novel effective feeder-free culture system for the Maintenance culture was carried out using the following: derivation of human induced pluripotent stem cells. Sci
  • NS / PCs neural stem / progenitor cells
  • Differentiated induction from human iPS cells to NS / PCs can be carried out according to known methods (Sugai K, Fukuzawa R, Shofuda T, Fukusumi H, Kawabata S, Nishiyama Y, Higuchi Y, Kawai K, Isoda M, Kanematsu D, Hashimoto-Tamaoki T, Kohyama J, Iwanami A, Suemizu H, Ikeda E, Matsumoto M, Kanemura Y, Nakamura M, Okano H.
  • EB embryoid body formation medium supplemented with 10 ⁇ M ROCK inhibitor Y276352 (10 ⁇ M SB431542, 100 nM LDN-193189 added to Stem Fit medium not supplemented with liquid C)
  • the cells were suspended, seeded at 9.0 ⁇ 10 3 cells / 75 ⁇ l per well of a low adhesion 96-well culture plate (Prime Surface 96V, Sumitomo Bakelite Co., Ltd.) and cultured.
  • 75 ⁇ l of EB formation medium was added, and thereafter, a half amount of EB formation medium was replaced every day and EB was obtained by culturing for 13 to 14 days.
  • EB is collected from each well, and 20 ng / ml recombinant human epidermal growth factor (PeproTech) is prepared in NS (neurosphere) medium (D-MEM / Ham's F-12 medium (containing HEPES) (Wako Pure Chemical Industries, Ltd.) (Wako Pure Chemical Industries, Ltd.) ), 20 ng / ml recombinant human fibroblast growth factor-2 (manufactured by PeproTech), 10 3 units / ml recombinant human leukemia inhibitory factor (manufactured by Nacalai Tesque), 2% B-27 supplement (Thermo Fisher Scientific) , And 1 unit / ml heparin sodium (manufactured by Ayway Pharma) was added for 7 days.
  • NS neurosphere
  • D-MEM / Ham's F-12 medium containing HEPES
  • WiproTech 20 ng / ml recombinant human fibroblast growth factor-2
  • the medium was changed once on the third or fourth day of culture.
  • the cell mass was collected by centrifugation, dispersed into single cells using TrypLE Select, and then suspended in NS medium.
  • the cells were seeded at a concentration of 1 ⁇ 10 5 cells / ml in a low adhesion flask (manufactured by Corning), the medium was changed every 3 to 4 days, and primary NS / PCs were obtained by culturing for 10 days.
  • primary NS / PCs are collected by centrifugation, dispersed into single cells using TrypLE Select, suspended in NS medium, and seeded at a concentration of 1 ⁇ 10 5 cells / ml in a low adhesion flask, The medium was changed every 3 to 4 days, and cultured for 7 to 10 days to obtain secondary, tertiary, quaternary, and quaternary NS / PCs.
  • yCD-UPRT expression lentiviral vector The cDNA of yCD (yest cytosine deaminase) -UPRT (uracil phosphoribosyl transferase) is pENTR / D-TOPO (PCR) from pSELECT-zeo-Fcy :: fur plasmid (manufactured by InvivoGen). The DNA was subcloned into Thermo Fisher Scientific Co., Ltd.) and the base sequence was confirmed.
  • the yCD-UPRT cDNA was subcloned into lentiviral vector plasmid CSIV-EF-RfA-IRES2-hKO1 to obtain CSIV-EF-yCD-UPRT-IRES2-hKO1.
  • CSIV-EF-yCD-UPRT-IRES2-hKO1 was used to construct a lentiviral vector that expresses yCD-UPRT and hKO1 (Humanized Kusabira-Orange fluorescent protein gene) under the EF-1 ⁇ promoter.
  • a lentiviral vector was prepared by a known method (Miyoshi H, Blomer U, Takahashi M, Gage FH, Verma IM. Development of a self-inactivating lentivirus vector. J Virol.
  • a CSIV-EF-yCD-UPRT-IRES2-hKO1 lentiviral vector is again infected at MOI 1-2, and stem cells for treating yCD-UPRT-expressing NS / PCs (TSC (1) ) was produced. It was confirmed that yCD-UPRT-expressing NS / PCs induce cell death by adding 1 ⁇ g / ml of 5-FC (Fluorocytosine) to the medium.
  • 5-FC Fluorocytosine
  • ⁇ YCD-UPRT Gene Transfer to GAPDH Gene Region by CRISPR / Cas9-Therapeutic Stem Cell TSC (2)> Preparation of iPS cells in which yCD-UPRT gene has been introduced into GAPDH gene region Using yap / CD9 gene editing technology, GAPDH and yCD-UPRT and Bsd are used to integrate yCD-UPRT into GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene region A construct for homologous recombination (HR-GAPDH-2A-yCD-UPRT-2A-Bsd) in which (blasticidin resistant gene) is inserted in a linked form at the 2A peptide sequence, and gRNA targeted near the stop codon of GAPDH And a Cas9 expression vector construct (U6-GAPDH-gRNA4-Cas9).
  • GAPDH and yCD-UPRT and Bsd are used to integrate yCD-UPRT into GAPDH (
  • constructs were introduced into a human iPS cell line (1210 B2) by electroporation, cultured in the presence of blasticidin S, and cloned. Confirmation of the homologous recombinant iPS cell line was performed by genomic PCR of each clone and nucleotide sequence confirmation by genomic sequencing.
  • ⁇ Therapeutic effect of yCD-UPRT expressing NS / PCs in brain tumor model mice Suicide gene cell therapy for malignant brain tumors using stem cells TSC (1) for yCD-UPRT expression and treatment (mixed transplantation model) T cells treated with general anesthesia using a mixture of U87-ffLuc 1 ⁇ 10 5 cells / 2 ⁇ l and yCD-UPRT-expressing TSC (1) (introducing yCD-UPRT to NS / PCs with lentiviral vector) 5 ⁇ 10 5 cells / 2 ⁇ l It transplanted stereotactically to the right striatum (2 mm right from Oizumi Gate, depth 3 mm from brain surface) of a deficient mouse (Female BALB / c nude mouse, 20 g, 6 w).
  • Serial changes of the tumor of the same individual were observed weekly with IVIS in vivo imaging system. IVIS during shooting, the VivoGlo TM Luciferin 30mg / ml under isoflurane inhalation anesthesia was administered 200 ⁇ l ip, it was taken at 10 minutes to reach the peak.
  • TSC stem cells
  • yCD-UPRT expression and treatment transplantation transplantation model
  • 1 x 10 6 cells / 2 ⁇ l of yCD-UPRT-expressing TSC (introduced yCD-UPRT into lentiviral vector into NS / PCs) were stereotactically transplanted to the same site 5 days later.
  • the tumor evaluation method, survival curve analysis, etc. were all performed in the same manner as the examination of the therapeutic effect of the above mixed transplantation model.
  • yCD-UPRT expression Visualization of bystander effect and migration of NS / PCs by slice culture using stem cell TSC (2) for therapy (time-lapse imaging)
  • Right striatum (2 mm right from Oizumi Gate, depth 3 mm from brain surface) of T cell deficient mice (Female BALB / c nude mouse, 20 g, 6 w) anesthetized with 1 ⁇ 10 5 U87-ffLuc mice.
  • Were transplanted stereotactically day 0.
  • yCD-UPRT-expressing TSC Introduced NS / PCs with yCD-UPRT with lentiviral vector
  • NS (neurosphere) medium D-MEM / Ham's F-12 medium (containing HEPES) (manufactured by Wako Pure Chemical Industries, Ltd.) (manufactured by Wako Pure Chemical Industries, Ltd.) 20 ng / ml recombinant human epidermal growth factor (manufactured by PeproTech) , 20 ng / ml recombinant human fibroblast growth factor-2 (PeproTech), 103 units / ml recombinant human leukemia inhibitory factor (Nakalai Tesque), 2% B-27 supplement (Thermo Fisher Scientific) ), Filled with 1 unit / ml heparin sodium (manufactured by Ayway Pharma).
  • D-MEM / Ham's F-12 medium containing HEPES
  • Wako Pure Chemical Industries, Ltd. 20 ng / ml recombinant human epidermal growth factor
  • PeproTech 20 ng / ml
  • the tumor was labeled green by Venus and red by TSC by hKO1, and from the same day, imaging was performed for 5 days with time-lapse under a confocal microscope.
  • the treatment medium was administered 200 ⁇ l of 10 mg / ml 5-FC in the above medium.
  • 200 ⁇ l of 1 M PBS was administered to the medium.
  • 250 ⁇ M of temozolomide was administered.
  • the medium was changed once every two days, and 5-FC, PBS and temozolomide were similarly administered.
  • TSC stem cells
  • yCD-UPRT expression and treatment transplantation transplantation model
  • Tumor evaluation methods and survival curve analysis were all performed as described above.
  • Tet Tetracycline induction to iPS cells using lentiviral vector
  • yCD-UPRT gene-Therapeutic stem cell TSC (3)> Preparation of Tet-inducible yCD-UPRT expression lentiviral vector
  • the yCD-UPRT cDNA was subcloned into lentiviral vector plasmid CSIV-RfA-TRE-EF-KT to obtain CSIV-yCD-UPRT-TRE-EF-KT.
  • CSIV-yCD-UPRT-TRE-EF-KT is a lentivirus in which hKO1 and rtTA (reverse Tet-controlled transactivator) are expressed under the EF-1 ⁇ promoter and yCD-UPRT is expressed under the tetracycline (Tet) inducible promoter It was used to create a vector. Lentiviral vectors were generated by known methods.
  • Tet-induced yCD-UPRT-expressing NS / PCs Human iPS cells are infected with a CSIV-yCD-UPRT-TRE-EF-KT lentiviral vector at an MOI of about 15, and after culture, clone sorting by a cell sorter is performed and hKO1 is stable Were obtained iPS cell lines that are highly expressed. It was confirmed that the addition of 1 ⁇ g / ml of doxycycline (Dox) and 1 ⁇ g / ml of 5-FC to the medium induces cell death in Tet-induced yCD-UPRT-expressing iPS cells.
  • Dox doxycycline
  • 5-FC 5-FC
  • Tet-induced yCD-UPRT-expressing iPS cells were induced to differentiate into NS / PCs to obtain secondary and tertiary Tet-induced yCD-UPRT-expressing NS / PCs. These Tet-induced yCD-UPRT-expressing NS / PCs were also confirmed to induce cell death by the addition of 1 ⁇ g / ml Dox and 1 ⁇ g / ml 5-FC to the medium.
  • TSC (3) Suicide gene cell therapy for malignant brain tumors using stem cells TSC (3) for treating Tet-induced yCD-UPRT expression (mixed transplantation model) T cell-deficient mouse (generally anesthetized) by mixing U87-ffLuc 1 ⁇ 10 5 cells / 2 ⁇ l and TSC (3) (introduce Tet induced yCD-UPRT into lPS cells with lentiviral vector) 5 ⁇ 10 5 cells / 2 ⁇ l It transplanted stereotactically to the right striatum (2 mm right from Oizumimon and depth 3 mm from brain surface) of Female BALB / c nude mouse, 20 g, 6 w.
  • Dox was ingested orally with 200 mg / kg of food. Tumor evaluation methods and survival curve analysis were all performed as described above.
  • CD suppresses DNA and RNA synthesis by converting 5-FC to 5-fluorouracil (5-FU) and induces cell death, but additionally by combining yCD with the uracil phosphoribosyltransferase (UPRT) gene Since UPRT converts 5-FU to 5-FUMP, which is a thymidylate synthase inhibitor, a strong bystander effect of 100 times or more is obtained as compared with yCD alone. Since the local bystander effect is cell cycle dependent and selectively kills only tumor cells, a high therapeutic index is expected.
  • UPRT uracil phosphoribosyltransferase
  • yCD-UPRT was transfected with a lentiviral vector to establish a therapeutic stem cell (TSC) expressing the suicide gene yCD-UPRT.
  • TSC therapeutic stem cell
  • T cell deficiency Mouse brain (right striatum) was transplanted with 1 ⁇ 10 4 human glioma cells (U87), and 5 days later, 1 ⁇ 10 6 TSC was stereotactically implanted at the same site.
  • TSC (+) / 5FC (-) 9).
  • TSC (+) / 5FC (+) 5 mg / day, n 3).
  • TSC TSC prepared by inducing differentiation of iPS cells into NC / PCs and gene transfer of yCD-UPRT with lentiviral vector
  • yCD-UPRT lentiviral vector
  • Tumors were labeled green by Venus and red by hKO1 and TSC was photographed for 5 days with a confocal microscope from the same day.
  • 200 ⁇ l of 10 mg / ml 5-FC was administered to the medium.
  • 200 ⁇ l of 1 M PBS was administered to the medium, and 250 ⁇ M temozolomide as a control group 2.
  • TSC TSC continued to survive, and it was possible to confirm migration and invasion to the inside of the tumor (Fig. 15, Fig. 16, Fig. 17).
  • temozolomide had an inhibitory effect on temporary tumor growth, but no reduction effect was observed (FIG. 15).
  • NS / PCs were induced from this iPS cell to obtain a therapeutic stem cell (TSC) expressing yCD-UPRT.
  • TSC therapeutic stem cell
  • T cell deficiency Mouse brain (right striatum) was transplanted with 1 ⁇ 10 4 human glioma cells (U87), and 5 days later, 1 ⁇ 10 6 TSC was stereotactically implanted at the same site.
  • TSC ( ⁇ ) / 5 FC (+), n 3)
  • tetracycline inducible yCD-UPRT is introduced into iPS cells by lentiviral vector and doxycycline (Dox) is added
  • Dox doxycycline
  • Dox was ingested orally with 200 mg / kg of food. Tumor evaluation methods and survival curve analysis were all performed as described above.
  • Tumors tended to shrink in the treatment group compared to both control groups (FIG. 25). Since the glioma cells (U87) were transfected with the fluorescent protein Venus, the tumor volume was quantitatively analyzed using a fluorescence microscope. Tumor volumes in the treatment group tended to shrink as compared to the control group (FIG. 26).
  • Example 2 Suicide gene cell therapy for glioma stem cells ⁇ expression / treatment of yCD-UPRT with CRISPR / Cas9, suicide gene cell therapy for malignant brain tumor stem cells (hG008) using stem cells (TSC) (post tumor formation transplantation model)> Human glioma stem cell line hG008-ffLuc 5 ⁇ 10 in the right striatum (2 mm right from Oizumi Gate, 3 mm depth from brain surface) of T-cell deficient mice (Female BALB / c nude mouse, 20 g, 6 w) under general anesthesia Five / two ⁇ l were transplanted, and after 45 days, 1 ⁇ 10 6 / two ⁇ l of yCD-UPRT-expressing TSC (introducing yCD-UPRT into CRISPR / Cas9 into iPS cells) was stereotactically implanted at the same site.
  • TSC stem cells
  • the tumor evaluation method, survival curve analysis, etc. were all performed in the same manner as the examination of the therapeutic effect of the above mixed transplantation model.
  • the present invention can be used in industrial fields such as pharmaceuticals.

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WO2020101570A1 (en) * 2018-11-14 2020-05-22 Advanced Cell Therapeutics Pte Ltd Stem cell infusion
WO2021201100A1 (ja) 2020-03-31 2021-10-07 学校法人 慶應義塾 ゲノム編集多能性幹細胞を用いた治療薬
CN115379845A (zh) * 2020-03-31 2022-11-22 学校法人庆应义塾 使用了基因组编辑多能干细胞的治疗药
EP4129311A4 (de) * 2020-03-31 2024-06-12 Keio University Therapeutikum mit genomeditierter pluripotenter stammzelle
CN115379845B (en) * 2020-03-31 2024-10-25 学校法人庆应义塾 Therapeutic agent using genome-editing pluripotent stem cells
CN112553286A (zh) * 2020-11-05 2021-03-26 北京大学深圳医院 自杀基因/前药系统疗效的评价方法和药物筛选方法

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